Miniaturized reaction vessel



Oct. 12, 1965 T. H. BENZINGER MINIATURIZED REACTION VESSEL Filed Jan.21, 1963 l o I: C v

JIIII III A INVENTOR.

Theodor H. Benzinger United States Patent 3,211,531 MINIATURIZEDREACTION VESSEL Theodor H. Benzinger, Chevy Chase, Md., assignor to theUnited States of America as represented by the Secretary of the NavyFiled Jan. 21, 1963, Ser. No. 253,008 8 Claims. (Cl. 225-259) (Grantedunder Title 35, US. Code (1952), see. 266) The invention describedherein may be manufactured and used by or for the Government of theUnited States of America for governmental purposes without the paymentof any royalties thereon or therefor.

The present invention relates generally to calorimetric apparatus andprocedures and, more particularly, to an improved reaction vessel forinvestigating the heat producing characteristics of reactions involvingmaterials in suspension and in solution.

In applicants copending application, Serial No. 17,232, filed March 23,1960, there is disclosed a heatburst microcalorimeter whose responsetime and sensitivity make it possible for the first time to investigatequantitatively certain chemical and biochemical reactions. In thisinstrument the reactants are widely dispersed so as to maximize the heattransfer surface area, and a so-called area thermopile, having its hotand cold junctions blanketing this surface and a confronting heat sinksurface, respectively, performs both as a low thermal impedance heattransfer means and a highly efiicient electrical signal generatingdevice. Because of the relationship and coaction between the reactionsurface, the thermopiles and the heat sink, the heat of reaction israpidly discharged into the heat sink as a heat pulse, and the passageof this pulse through the thermopile imparts to the output voltagesignal a pulse configuration. As a consequence of this mode ofoperation, which for the first time fully exploits the thermal potentialdifference between the reaction and the heat sink, the microcalorimeterof the above application has an increased sensitivity to instantaneousreactions, .an improved speed of response to changing continuous rate ofheat flow and a reduced error from inertial distortions and externaltemperature disturbances.

Moreover, to eliminate the need for mechanical mixing and stirringdevices, a source of unknown error in the prior art equipment, theheatburst microcalon'meter of the above application and its reactionvessels are constructed and mounted so that only gravitational forcesare used for carrying out these operations. Because of the enhancedpower of resolution of this instrument, even the relatively small amountof heat introduced by this method of mixing can be accuratelyascertained by repeating the original mixing motions after thermalequilibrium has been established and recording the voltage wave form sogenerated.

One of the major factors influencing the construction of the reactionvessel and the blank vessel comes from the previous stated desideratumthat the reaction material present the largest obtainable surface areato the area thermopile for accelerating the heat flow therefrom into theheat sink. To achieve this dispersal, applicants copending application,Serial No. 17,232, employs reaction vessels wherein the bulk material isaccommodated between concentric, cylindrical walls, with the otherreactants isolated therefrom prior to mixing by either a longitudinaldividing wall, a circular transverse wall, drop wells or ring-typedropholders. However, in these vessels reactants cannot be keptseparated during rotating motion. Therefore, for experiments involvingliving material, such as, for example, bacteria or cell cultures, a testtube type reaction vessel for continuous rotation,

closed ofi at each end by a cap having a dome-shaped compartmentprojecting from the inner wall thereof, is utilized. Each of thesecompartments has an aperture formed at its apex. With this design,different substances can be accommodated in each compartment, and thesesubstances can be kept separated during rotating motions and yet bemixed, though only incompletely, at predetermined times with the bulksuspension by tilting. Thus, one holder may, for example, add virus to acell culture while the other may add an enzyme poison to stop allmetabolic activity and establish the zero base level of the system atthe end of a particular recording, rotating operation.

Most biological materials, with the exceptions of solutions of molecularparticles, consist of matter in suspension rather than in solution. Inthis category are, for example, microorganisms, parasites, protozoa,eggs, sperms, bacteria, virus, tissue slices, cells, cell cultures,homogenates, subcellular particles, such as nuclei, spindles,mitochondria, microsomes. With many of these objects, the heatproduction and oxygen consumption depend upon the rate of diffusion ofoxygen to the often sedimented and tightly packed particles.Consequently, the reaction vessels accommodating these objects must berotated during the investigation. Because :of this mode of operation,some provision must be made in the vessel for keeping two or threereactants separated during periods of such continuous rotation.

With some of the reaction vessels disclosed in copending application,Serial No. 17,232, some difliculty has been experienced in rinsing outthe reactants and insuring that each drop thereof mixes with the bulksolution accommodated in the main body portion of the vessel. As aresult, these substances have to be inserted stoichiometrically inexcess of the bulk solution. This, of course, complicates the datareduction operation.

These difiiculties are overcome by the invention which is based on adiscovery used also in copending application, Serial No. 253,007, filedJanuary 21, 1963, for the miniaturized reaction vessel, namely, thatliquid materials flow easily in, and may be easily rinsed out from,narrow annular spaces between two cylindrical surfaces.

It is accordingly a primary object of the present invention to providean improved reaction vessel for a rotating microcalorimetric apparatusand for procedures with materials in suspension rather than solution.

Another object of the present invention is to provide a reaction vesselfor microcalorimetry which accommodates a bulk solution of one andsmaller quantities of two other, difierent reactants.

A still further object of the present invention is to provide a reactionvessel wherein certain reactants are accommodated in annular spacesbetween two cylindrical wal s.

A yet still further object of the present invention is to provide areaction vessel wherein various reactants are kept separate fromsuspended material during rotation and are mixed therewith upon tilting.

A yet still further object of the present invention is to provide animproved reaction vessel wherein the reactants are accommodated within acombination cap and dropholder.

A still further object of the present invention is to provide a reactionvessel of simple design which is easy to assemble, disassemble andclean.

Other objects and many of the attendant advantages of this inventionwill be readily appreciated as the same becomes better understood byreference to the following detailed description when considered inconnection with the accompanying drawings wherein:

FIG. 1 illustrates a heatburst microcalorimeter of the 3 type disclosedin applicants copending application, Serial No. 17,232; and

FIG. 2 is one embodiment of a reaction vessel constructed according tothe present invention.

Referring now to FIG. 1 of the drawings, the heatburst microcalorimeterin the twin calorimeter configuration is seen to comprise adou'blewalled reaction vessel 20 and a blank vessel 21 disposed in anend-to-end relationship within a cylindrical heat conducting sleevewhose outer surface is completely blanketed by the hot junctions of apair of area thermopiles 22 and 23. Each of these so-called areathermopiles is fabricated by first winding a constantan conductor in ahelix, then copperplating half of each individual turn thereof to formthermojunctions at diametrically opposite points, and thereafter coilingthe plated helical structure about the outer surface of the cylindricalsleeve. Surrounding both area thermopiles is a cylindrical heat sink 24whose inner surface is completely blanketed with the cold junctions ofthese thermopiles. This heat sink is closed off by metal covers 26 andalso by heavy metallic caps 27, the latter serving to increase the heatcapacity of the system and provide a thermal short circuit between thevarious arcuated sectors forming the heat sink. The above assembly,which is isolated from the local environment by a pair of confronting U-shaped De War vessels 35, is held in radial suspension to furtherrestrict heat inroads into the temperature sensitive portion of theinstrument by means of wires 29 which are fastened to ring 28 and heatsink 24. Also secured to this suspension ring are a pair of closuremembers 38, and the space between these members and the De War vesselsis filled with an insulating material, such as Styrofoam, for additionalthermal protection. A suitable pair of shafts 39 attached to oppositeends of these closure members permit the complete instrument to berotated about its longitudinal axis for mixing and other allied purposessuch as preventing sedimentation of suspended particles.

In investigating the possible use of capillary devices for retainingquantities of reactant, it was discovered that liquids and gases tend toflow more easily in narrow, annular spaces between cylindrical surfacesthan in open capillaries. For example, the flow in a one millimeterannular space was found to be better than that in an open capillary ofthree millimeter diameter. For best flow results, it was experimentallydetermined that the inner diameter of the annulus should preferablyexceed 1.25 mm.

Referring now to FIG. 2, it will be seen that the reaction vessel of thepresent invention embodying this discovery, in one modification,consists of a test-tubelike tubular member 50, closed off at each endwith combination cap and dropholders 51 and 52. Each cap has an end wallportion 53 and a circular flange 54 which is adapted to fit over thereduced diameter end portion 55 and form an airtight seal therewith. Byreducing the diameter of member St) at each end by approximately thethickness of this flange, the assembled vessel presents anuninterrupted, cylindrical shape to the cylindrical sleeve of thecalorimeter into which it fits. Matching holes 56 and 57 are drilledthrough both caps and the reduced diameter portion of member 50. Theapertures permit air to escape from the reaction vessel when theapparatus is assembled. Also, these apertures provide access to theinterior of the vessel so that, for example, the nose of a conventionalfluid dropper can be inserted therethrough during the bulk fluid fillingoperation.

Supported from the iner wall of each cap by a stem or stalk 58 is acapillary or tubular element 59 which has concentrically disposedtherein a cylindrical core 60. This core extends a slight distancebeyond the top rim of capillary 59 and is rounded off to prevent any ofthe reactant from adhering thereto. From what has been saidhereinbefore, it will be appreciated that the annular space 61 definedby the inner wall of capillary 59 and the outer wall of core 60 servesas the temporary retainer for the reactant.

When measured droplets of reactants are placed in these annular spaces,capillary action prevents the material from flowing out even when thevessel is tilted into a perpendicular position. As long as the reactantvessel is kept in a more or less horizontal position when filled, thebulk liquid 62, with suspended material in its main space, will remainisolated from the various reactants. And this isolation of the firstreactant persists even when the vessel is rotated abouts itslongitudinal axis. However, when the vessel is tilted upwardly, forexample, the second reactant in the lower well will be almostinstantaneously washed out as the bulk liquid first splashes against thelower end wall and then rises through the annular space between thecapillary and its central core. Likewise, and first when the vessel istilted in an opposite direction, the bulk liquid will wash out the thirdreactant housed within the opposite well.

It will be appreciated that the stalks or stems supporting the capillarytubes should be of minimum thickness so as not to interfere too muchwith the entrance of the liquid into the annular space.

Since the apparatus of FIG. 2 comes apart easily at the end caps, theassembly, disassembly and cleaning of the vessel present no problems.Likewise, since the filling of the wells is done with the caps detached,this operation can be performed without difliculty and with a highdegree of precision.

To prepare the apparatus for use, each of the annular spaces is filledby a fluid dropper. The closure caps are then fitted to the open ends ofthe reaction vessel and rotated to align the matching holes 56 and 57.One of these apertures may now be employed as a filling aperture and aneedle-nose dropper inserted therethrough into the main body of thereaction vessel for introducing the bulk fluid 62. These holes canthereafter be closed by simply twisting each cap in its cylindricalgrease joint and, after thermal equilibrium has been obtained, thereaction vessel may be placed within the core of the calorimeter.

The vessels constructed according to the modification of FIG. 2 may befabricated to accommodate approximately four milliliters of bulksolution and one hundred to two hundred microliters of reactants in thewells. They may be made of glass with carbon molding techniques to holdthe tolerances of the cap joints, or of gold-laminated stainless steelwith solid gold caps. And by designing them With a relatively smalldiameter, for example, approximately fifteen millimeters, the end lossesof heat men tioned above can be reduced to an acceptable level.

While the concept of temporarily storing relatively small quantities offluids or materials in annular spaces and washing them out by or mixingthem with another fluid which is caused to flow through these spaces isdisclosed in connection with calorimetry instruments and the like, itwill, of course, be appreciated that this technique is of a genericnature and may be used in other chemical or fluid mixing and stirringapplications.

It would also be mentioned that while the capillary 59 and its core 60are mounted along the axis of symmetry of tubular element 50, thesestructures can, of course, be displaced from this location to suit theparticular conditrons prevailing. Also, the cross-sectional shape of thecapillary and its core can take various forms other than thatillustrated in FIG. 2.

It would also be pointed out that the reaction vessel can be formed withan integral closure piece so that only one end cap need be used. Ofcourse, this would mean that only one reaction substance would beavailable to coact with the bulk solution. It would also be pointed outthat in the modification of FIG. 2 the bulk solution is placed in themain body of the vessel only after both of the end caps are in place forobvious reasons. Because of the reduced diameter portions of thereaction vessel adjacent each end thereof, a shallow Well is created inthe main body of the tube, and this well can accommodate a finite amountof bulk fluid. Thus, if desired, the bulk fluid can be introduced intothis well when the vessel is in a horizontal attitude with either orboth end caps removed.

Obviously many modifications and variations of the present invention arepossible in the light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

What is claimed is:

1. For use with a reaction vessel having one end open, the combinationof a closure cap having an end wall portion,

a sleeve supported from said end wall and spaced therefrom,

said sleeve occupying a position within the main body of said reactionvessel when said closure cap is secured to the open end of said reactionvessel,

and a rod retained within said sleeve with its longitudinal axisparallel to the axis of symmetry of said sleeve,

the separation between the inner wall surface of said sleeve and theouter Wall surface of said rod being such that a fluid substance placedtherebetween is retained therein by capillary action.

2. In an arrangement as defined in claim 1 wherein said rod isconcentrically positioned within said sleeve and extends the completelength thereof.

3. In an arrangement as defined in claim 2 wherein said rod is supportedfrom the end wall portion of said closure cap.

4. A combined closure cap and interim fluid storage device for anopen-ended reaction vessel comprising,

a member having an end wall portion and a rim portion projectingtherefrom,

a sleeve supported away from said end wall portion with its longitudinalaxis perpendicular to said end wall portion,

a core supported from said end Wall and extending the length of saidsleeve,

the separation between the inner wall surface of said sleeve and theouter surface of said core being such that a fluid substance disposedtherebetween is maintained therein by capillary action.

5. In combination, a tubular element,

said tubular element having a reduced outer diameter portion adjacenteach end thereof,

a removable closure cap sealing off each open end of said tubularelement and making said tubular element fluidtight,

each closure cap having an end wall portion and a circular rim portion,

the inner diameter of said rim portion being slightly greater than theouter diameter of the reduced diameter portion of said tubular element,

and the outer diameter of said rim portion being equal to the unreducedouter diameter of said tubular element.

each rim portion and each reduced diameter portion having an aperturecut therethrough,

the aperture in each rim portion and reduced diameter portion beingaligned when said closure cap is in place,

a sleeve secured to the end wall portion of each closure cap in a spacedrelationship therefrom,

10 a core retained within each sleeve, the longitudinal axis of eachcore coinciding with the longitudinal axis of symmetry of each sleeve.

6. A reaction vessel for use in the core of a calorimeter of the typewherein only gravitational forces are employed to intermix the reactantscomprising a tubular member;

means closing off the ends of said member and forming with said member afluidtight enclosure;

a sleeve;

means for supporting said sleeve within said fluidtight enclosure suchthat a flow of fluid can take place into one end of said sleeve, throughsaid sleeve and out the other end thereof; core concentrically retainedWithin said sleeve, the space between said core and the inner wall ofsaid sleeve serving as an open-ended annular compartment for retainingtherein by capillary action a fluid reactant.

7. A reaction vessel for use in the core of a calorimeter of the typewherein only gravitational forces are employed to intermix the reactantscomprising a tubular member;

a detachable closure element sealing off each end of said member andforming with said member a fluidtight enclosure;

a pair of sleeves;

means connected to each closure element for supporting one sleeve ofsaid pair of sleeves Within said fluidtight enclosure such that a flowof fluid can take place into one end of that sleeve, through that sleeveand out the other end thereof; and

a core concentrically disposed within each sleeve, the space between theinner wall surface of each sleeve and each core being such that anyreactant fluid placed therein is retained there by capillary action.

8. In an arrangement as defined in claim 7 wherein each core consists ofa cylindrical member which has a diameter of at least 1.25 millimeters.

References Cited by the Examiner UNITED STATES PATENTS 1,482,966 2/24Bevan 23-253 1,594,370 8/26 Kubota 23--253 2,052,185 8/36 Lewis 73-382MORRIS O. WOLK, Primary Examiner.

JAMES H. TAYMAN, JR., Examiner.

1. FOR USE WITH A REACTION VESSEL HAVING ONE END OPEN, THE COMBINATIONOF A CLOSURE CAP HAVING AN END WALL PROTION, A SLEEVE SUPPORTED FROMSAID END WALL AND SPACED THEREFROM, SAID SLEEVE OCCUPYING A POSITIONWITHIN THE MAIN BODY OF SAID REACTION VESSEL WHEN SAID CLOSURE CAP ISSECURED TO THE OPEN END OF SAID REACTION VESSEL, AND A ROD RETAINEDWITHIN SAID SLEEVE WITH ITS LONGITUDINAL AXIS PARALLEL TO THE AXIS OFSYMMETRY OF SAID SLEEVE, THE SEPARATION BETWEEN THE INNER WALL SURFACEOF SAID SLEEVE AND THE OUTER WALL SURFACE OF SAID ROD BEING SUCH THAT AFLUID SUBSTANCE PLACED THEREBETWEEN IS RETAINED THEREIN BY CAPILLARYACTION.